PROJECT SUMMARY Liver fibrosis, and ultimately cirrhosis, is the final common pathway of chronic liver diseases induced by any etiology. Liver failure due to cirrhosis is the 12th leading cause of death by disease in the United States and there is no effective current treatment other than liver transplantation. One hallmark of liver cirrhosis is that the liver extracellular matrix becomes stiffer, but how the stiffened microenvironment causes hepatocyte dysfunction is not completely understood. Our proposed research addresses this gap in knowledge and is designed to deliver data that will lead to tangible advances in the treatment of liver cirrhosis, which may include development of 1) novel therapies that maintain adequate liver function in patients with progressive fibrotic liver disease, 2) clinical prognostic models that predict which patients with resolving fibrosis may regain sufficient hepatic function, and 3) highly functional tissue-engineered liver constructs for tissue replacement therapy in patients with end-stage liver insufficiency. Our preliminary studies show that hepatocytes are exquisitely responsive to the mechanical cues of extracellular matrix tuned to the stiffness of fibrotic livers, and that the induced downstream signaling pathways (i.e. mechanotransduction) directly inhibit hepatocyte function. Using a multi-disciplinary cross-modality approach, we propose to further delineate the mechanism of how a stiffened microenvironment induces hepatocyte dysfunction and its clinical applicability. These scientific objectives will be achieved first, by determining the key molecular players that translate mechanical cues into intracellular signals that inhibit hepatocyte function using genetically engineered mouse models with tissue-specific and temporally-induced expression of key mechanotransduction molecules. Subsequently, we propose to verify the clinical relevance of these molecular mechanisms by characterizing matrix rigidity at the microscale level in normal and cirrhotic human livers as determined by atomic force microscopy, in conjunction with single-cell gene expression analysis. Finally, we propose to test whether the relationship between microenvironment rigidity and hepatocyte function may be recapitulated in complex tissue-engineered liver constructs that are produced by three-dimensional bioprinting ex vivo and tuned to the stiffness of normal or fibrotic human liver. The proposed research is conceptually innovative because, instead of attempting to therapeutically target the process of fibrosis, we aim to understand the hepatocyte response to fibrosis, thereby prompting new approaches to treat and prognosticate chronic liver disease that focus on modulating the hepatocyte response to the fibrotic stimulus. It is, in the end, liver functional failure that causes death from liver disease, and not necessarily the process of fibrosis in itself. Results from this proposed study will authoritatively define the role of matrix rigidity in modulating hepatocyte function and illuminate several paths toward clinical translation.